Our studies (Infect. Immun. 73:1679-84, 2005) have recently demonstrated that N. gonorrhoeas can form a biofilm. Studies show that gonococci can form biofilms in the presence and absence of CMP-Neu5ac. Light microscopic analysis of frozen sections of biofilm showed evidence of organisms embedded in a matrix, which was interlaced with water channels. Cryo-field SEM and TEM confirmed that organisms were embedded in a continuous matrix with membrane structures covering and spanning the biofilm. Since the last submission of this proposal, we have generated immunohistochemical data that supports the hypothesis that gonococci may be forming biofilms during natural cervical infection. We are proposing to expand on this data in the revised grant. In addition, we have extensively studied six gonococcal wildtype strains and 12 different mutants in strain 1291 for the ability to form biofilms in a flow chamber and over cervical epithelial cells under both aerobic and anaerobic conditions. All of the wildtype gonococci were able to form biofilms. There were differences in the ability of the mutants to form biofilms under different environmental conditions. We have performed extensive SEM and TEM analysis on biofilms grown in flow chambers and over epithelial cells. These studies suggest that the organism forms a membranous network that appears to act as a scaffold for the biofilm. Since these membranes can be of a size (20 to 30 microns in length) that precludes them being merely the product of bacterial cell lysis, we believe that they are primarily the result of fusion of the extensive gonococcal membrane blebs which form during biofilm growth. We would hypothesize that colonization of the female by gonococci appears to involve two distinct environmental niches, a biofilm and an intracellular state. In this proposal, we hypothesize that 1) N. gonorrhoeae produces a biofilm, 2) that this biofilm is a complex structure involving assembly of large membranous structures as well as a matrix most probably derived from outer membrane blebs, and 3) that the pattern of gonococcal gene and protein expression is different in the biofilm phase compared to the planktonic phase of growth. These hypotheses will be resolved by the following specific aims;1) Determination of the structure of the gonococcal biofilm grown in flow chambers and over human cervical epithelial cells, 2) Comparative global analysis of the transcriptome of N. gonorrhoeae in the planktonic and biofilm phases in continuous flow chambers and over primary human cervical epithelial cells and 3) comparative global analysis of the proteome of N. gonorrhoeae in the planktonic and biofilm phases in continuous flow chambers and over primary human cervical epithelial cells. This will be accomplished by studying the subproteomes of the cytoplasmic fraction, inner and outer membranes.